Previously, possible interaction between the opioid peptide derived from proenkephalin met-enkephalin-arg-phe (YGGFMRF) and phe-met-arg-phe-NH2 (FMRF-NH2) was investigated. FMRF-NH2 injected intrathecally reduced the analgesia induced by YGGFMRF. Though FMRF-NH2 immunoreactive material (FMRF-NH2-IR) is present in rat spinal cords, it is different from FMRF-NH2. Because of this, endogenous FMRF-NH2-IR was isolated from the medulla oblongata of bovine brain by immunoaffinity column chromatography using IgG from FMRF-NH2 antiserum conjugated to Sepharose 4B as affinity ligand. The partially purified material, which contained mainly 2 immunoreactive peptides with molecular weight of about 1500-1800 daltons, attenuated the analgesia induced by YGGFMRF when injected intrathecally. The results suggest a naloxone like activity for FMRF-NH2 or endogenous FMRF-NH2-IR. Physiological role of endogenous FMRF-NH2-IR was assessed using IgG from FMRF-NH2 antiserum as antagonist. IgG from FMRF-NH2 antiserum caused a moderate long lasting naloxone reversible analgesia. This result implies a modulatory role for FMRF-NH2-IR in analgesia possibly mediated by endogenous opioid peptide. FMRF-NH2-IR is unevenly distributed in the rat brain with high coincentrations in pituitary and hypothalamus and low levels in hippocampus and cerebellum. Biochemical analysis revealed three different forms of FMRF-NH2-IR in the rat brain and, interestingly, the proportion of the 3 FMRF-NH2-IR varies greatly in different regions of rat brain. Whether all three different forms have similar biological activities still remains to be established.